RESUMEN

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Asunto(s)

Progresión de la Enfermedad , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacología , Melanoma Experimental/patología , Imitación Molecular , Metástasis de la Neoplasia , Animales , Línea Celular Tumoral , Citometría de Flujo , Melanoma Experimental/irrigación sanguínea , Ratones , Resonancia por Plasmón de Superficie

RESUMEN

The initial choice of anticoagulant therapy administered in emergency departments for acute coronary syndromes (ACS) has important consequences for subsequent patient care, as neither unfractionated heparin (UFH) nor low-molecular-weight heparin (LMWH) are ideally suited for all potential clinical treatment pathways. UFH remains widely used for surgical interventions because of the ability to rapidly reverse its anticoagulant activity. However, the unpredictable pharmacokinetic profile of UFH presents safety issues, and the low subcutaneous bioavailability limits the utility of UFH for patients who are medically managed. LMWH has superior pharmacokinetic properties, but its anticoagulant activity cannot be effectively monitored or reversed during surgery. There is an unmet medical need for a baseline anticoagulant therapy that addresses these shortcomings while retaining the beneficial properties of both UFH and LMWH. We describe here M118, a novel LMWH designed specifically for use in the treatment of ACS. M118 shows broad anticoagulant activity, including potent activity against both factor Xa (~240 IU/mg) and thrombin (factor IIa; ~170 IU/mg), low polydispersity, high (78%) subcutaneous bioavailability in rabbits, and predictable subcutaneous and intravenous pharmacokinetics. Additionally, the anticoagulant activity of M118 is monitorable by standard coagulation assays and is reversible with protamine. M118 demonstrates superior activity to conventional LMWH in a rabbit model of abdominal arterial thrombosis without increasing bleeding risk, and is currently being evaluated in a phase II clinical trial evaluating efficacy and safety in patients undergoing percutaneous coronary intervention.

Asunto(s)

Síndrome Coronario Agudo/tratamiento farmacológico , Anticoagulantes/uso terapéutico , Enfermedades de la Aorta/tratamiento farmacológico , Arteriopatías Oclusivas/tratamiento farmacológico , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Anticoagulantes/toxicidad , Aorta Abdominal , Disponibilidad Biológica , Modelos Animales de Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Inhibidores del Factor Xa , Hemorragia/inducido químicamente , Heparina de Bajo-Peso-Molecular/administración & dosificación , Heparina de Bajo-Peso-Molecular/química , Heparina de Bajo-Peso-Molecular/aislamiento & purificación , Heparina de Bajo-Peso-Molecular/uso terapéutico , Heparina de Bajo-Peso-Molecular/toxicidad , Inyecciones Intravenosas , Inyecciones Subcutáneas , Mucosa Intestinal/química , Masculino , Conejos , Porcinos , Trombina/antagonistas & inhibidores

RESUMEN

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/fisiología , Heparitina Sulfato/metabolismo , Neoplasias Pulmonares/prevención & control , Melanoma Experimental/prevención & control , Neurofibromina 2/genética , Animales , Western Blotting , Adhesión Celular , Ensayo de Unidades Formadoras de Colonias , Neoplasias Pulmonares/secundario , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Transfección , Células Tumorales Cultivadas

RESUMEN

Melanoma B16-F10 cells and Lewis lung carcinoma LL/2 cells were engineered with a bacterial gene -- chloramphenicol acetyl transferase (CAT) -- by establishing stable transductants. Expression of CAT in both cell types did not alter the ability of these cells to grow into tumors when injected subcutaneously into mice. In addition, the measurement of CAT levels in the lung using a simple ELISA assay revealed a close correlation with direct counting of metastatic nodules. Thus, the CAT-expressing cells will likely have wide ranging applications to quantify tumor metastasis especially in situations where visual counting is difficult. The availability of genetically labeled mouse B16-F10 melanoma and Lewis lung carcinoma cell lines will facilitate future studies of the mechanism and progression of cancer and the discovery of new therapies.

Asunto(s)

Carcinoma Pulmonar de Lewis/patología , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Modelos Animales de Enfermedad , Ingeniería Genética , Melanoma/genética , Melanoma/veterinaria , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/veterinaria , Animales , Bacterias/genética , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Ratones , Transducción Genética , Células Tumorales Cultivadas